The introduction of exogenous genetic material (transgene) or foreign molecules in living cells has been found, during the last decades, to be a powerful tool in modern molecular biology. In the case of incorporation of exogenous DNA molecules in eukaryotic cells, usually mammals, it is called transfection, which may be transient or stable. More generally, the interest is for the incorporation of different materials foreign to the cell, such as imaging agents, peptides, antibodies, enzymes or pharmacologically active molecules.
Several techniques have been developed to introduce exogenous material in the cytoplasm or in the nucleus of a eukaryotic cell, among these chemical methods, viral methods and physical methods. The eukaryotic cell is enclosed in a cell (or plasma) membrane that delimits it and has a typical thickness of about 5 nm. The introduction of exogenous material through physical methods takes place by the passage through the cell membrane.
Physical methods often used are electroporation and microinjection. In electroporation, the cells are immersed into a solution containing the DNA or the molecules to be introduced and are subjected to short and intense electric pulses that produce transient pores in the cell membrane through which the exogenous material can enter. Microinjection consists in introducing the material directly into the nucleus or in the cytoplasm of the cell using a thin needle attached to a microinjector.
Effective insertion techniques that can be used on the vast majority of cell types use a laser beam to create a localized transient membrane rupture. In particular, in optoinjection (or optical injection), high-intensity laser pulses generate transient pores in the membrane which allow the influx of extracellular material within the cell.
An overview of the physical methods is given by Meacham J. M. et al. in “Physical methods for intracellular delivery: practical aspects from laboratory use to industrial-scale processing” published in Journal of Laboratory Automation, vol. 19 (2014), pages 1-18.
The cell membrane is a thin layer of hydrophobic lipids soaked in protein molecules which cross the membrane or are positioned on the inner or outer surface thereof. The cell's lipid nature makes sure that, under normal conditions, the membrane acts as a waterproof barrier to the passage of most water-soluble molecules. In techniques that use the interaction of a laser beam with the cell, obtaining an accurate alignment and positioning of the laser beam focus on the thin membrane is often a difficult and time-consuming task.
There are several known mechanisms that enable the transient perforation of cells, including methods that take advantage of a two-photon process using a femtosecond laser as the optical source. In “Femtosecond cellular transfection using a nondiffracting light beam” by X. Tsampoula et al., published in Applied Physics Letters 91, 053902 (2007), the authors use a Bessel beam to obviate the need to precisely locate the laser focus on the cell membrane, allowing the two-photon excitation along a line leading to the cell transfection.
Stevenson D. et al. in “Femtosecond optical transfection of cells: viability and efficiency”, Optics Express, vol. 14, no. 16, page 7125, present a femtosecond optical transfection efficiency analysis using an 800 nm titanium sapphire laser.
A method of optical injection uses a nano- or microparticle optically trapped on the surface of a cell to be injected into the cell. In “Targeted optical injection of gold nanoparticles into single mammalian cells”, C. McDougall et al., in J. Biophotonics 2, 736-743 (2009), the authors study an optical technique for inserting a nanoparticle of gold of 100 nm in diameter within a single cell by combining optical trapping and optical injection. The described optical apparatus includes a femtosecond laser for optical injection and a continuous wave laser for optical trapping. The positioning of the laser beam focus was obtained through an xyz translation system on which the sample dish was placed. According to the authors, the pulsed source causes a transient optical force on a nanoparticle and this has been believed to promote the entry of the nanoparticle through the cell membrane.
Patent application WO 2011/124899 relates to a method for poration of the membrane of biological cells in a specific area and the large scale poration with microbubbles excited by the rupture induced by laser light of single optically trapped nanoparticles.
Waleed et al. in “Single-cell opto-poration and transfection using femtosecond laser and optical tweezers”, Biomedical Optics Express Vol. 4, No. 9 (2013), describe a transfection technique which involves trapping and inserting a plasmid-coated polystyrene microparticle in an MCF-7 cell. The cell membrane is first pierced and then the microparticle is inserted into the cell using optical tweezers. Three laser beams are used: the first beam is a femtosecond laser at 800 nm to pierce the membrane; the second is a continuous wave (CW) laser at 1064 nm, whose function is to trap and insert the microparticle in the cell, while the third laser beam is a CW laser at 685 nm, which senses the exact position of the membrane so that the 1064 nm entrapping laser can introduce the microparticle through the pierced hole in the cell membrane.
Antkowiak et al. in “Application of dynamic diffractive optics for enhanced femtosecond laser based cell transfection”, J. Biophotonics 3, No. 10-11, 696-705 (2010), describes the use of a spatial light modulator (SLM) which acts as a dynamic diffractive optical element, which provides a lateral and axial beam control. The authors studied the feasibility of applying radiative doses at various axial and lateral positions using the SLM. In one method, the beam is focused sequentially in three different axial positions separated by 1 μm with a 700 ms delay between consecutive doses, which the authors say is a delay long enough to avoid any accumulation process of the photon energy. The authors conclude that the radiation in three axial positions doubles the number of cells actually optoinjected compared to a single dose.